Peptides and peptidomimetics with structural similarity to human P53 that activate P53 function

ABSTRACT

The present invention provides peptides and peptidomimetics corresponding to part or to the entirety of the region encompassed by residues 360-386 of human p53, said peptides and peptidomimetics characterized by the ability to activate DNA binding of wild-type p53 and to select tumor-derived p53 mutants. Pharmaceutical compositions of the compounds of the invention and methods of using these compositions therapeutically are also provided.

This application is a 371 of PCT/US96/01535, filed Feb. 12, 1996, and acontinuation-in-part of application Ser. No. 08/392542, filed Feb. 16,1995, now U.S. Pat. No. 6,169,073.

FIELD OF THE INVENTION

The present invention relates to the field of peptides derived fromtumor suppressor proteins and their use in therapy and drug design.

BACKGROUND OF TIE INVENTION

Wild-type (wt) p53 is a sequence-specific DNA binding protein found inhumans and other mammals, which has tumor suppressor function [Harris(1993), Science, 262: 1980-1981]. The gene encoding p53 is mutated inmore than half of all human tumors, suggesting that inactivation of thefunction of the p53 protein is critical for tumor development.

The nucleotide sequence of the human p53 gene and the amino acidsequence of the encoded p53 protein have been reported [Zakut-Houri etal. (1985), EMBO J., 4: 1251-1255; GenBank Code Hsp53]. These sequencesare presented below as SEQ ID NOs: 1 and 2, respectively. The amino acidsequence of p53 is conserved across evolution [Soussi et a]. (1990),Oncogene, 5: 945-952], suggesting that its function is also conserved.

The p53 protein functions to regulate cell proliferation and cell death(also known as apoptosis). It also participates in the response of thecell to DNA damaging agents [Harris (1993), cited above]. Thesefunctions require that p53 binds DNA in a sequence-specific manner andsubsequently activates transcription [Pietenpol et al. (1994), Proc.Natl. Acad. Sci. USA, 91: 1998-2002]. References herein to DNA bindingactivity of p53 are concerned with this sequence-specific binding unlessotherwise indicated.

In more than half of all human tumors, the gene encoding p53 is mutated[Harris (1993), cited above]. Thus, the encoded mutant p53 protein isunable to bind DNA [Bargonetti et al. (1992), Genes Dev., 6: 1886-1898]and perform its tumor suppressing function. The loss of p53 function iscritical for tumor development. Introduction of wild-type p53 into tumorcells leads to arrest of cell proliferation or cell death [Finlay et al.(1989), Cell, 57: 1083-1093; Eliyahu et al. (1989), Proc. Natl. Acad.Sci. USA, 86: 8763-8767; Baker et al. (1990), Science, 249: 912-915;Mercer et al. (1990), Proc. Natl. Acad. Sci. USA, 87: 6166-6170; Dilleret al. (1990), Mol. Cell. Biol., 10: 5772-5781; Isaacs et al. (1991),Cancer Res., 51: 4716-4720; Yonish-Rouach et al. (1993), Mol. Cell.Biol., 13: 1415-1423; Lowe et al. (1993), Cell, 74: 957-967; Fujiwara etal. (1993), Cancer Res., 53: 4129-4133; Fujiwara et al. (1994), CancerRes., 54: 2287-2291]. Thus, if it were possible to activate DNA bindingof tumor-derived p53 mutant proteins, then tumor growth would bearrested. Even for tumors that express wild-type p53., activation of itsDNA binding activity might arrest tumor growth by potentiating thefunction of the endogenous p53 protein.

The N-terminus of p53 (residues 1-90 of the wild-type p53 sequencestored under GenBank Code Hsp53 and repeated here as SEQ ID NO:2; allresidue numbers reported herein correspond to this sequence) encodes itstranscription activation domain, also known as transactivation domain[Fields et al. (1990), Science, 249: 1046-1049]. The sequence-specificDNA binding domain has been mapped to amino acid residues 90-289 ofwild-type p53 [Halazonetis and Kandil (1993), EMBO J., 12: 5057-5064;Pavletich et al. (1993), Genes Dev., 7:2556-2564; Wang et al. (1993),Genes Dev., 7:2575-2586]. C-terminal to the DNA binding domain, p53contains a tetramerization domain. This domain maps to residues 322-355of p53 [Wang et al. (1994), Mol. Cell. Biol., 14: 5182-5191]. Throughthe action of this domain p53 forms homotetramers and maintains itstetrameric stoichiometry even when bound to DNA [Kraiss et al. (1988),J. Virol., 62: 4737-4744; Stenger et al. (1992), Mol. Carcinog.,5:102-106; Sturzbecher et al. (1992), Oncogene, 7: 1513-1523; Friedmanet al. (1993), Proc. Natl. Acad. Sci. USA, 90: 3319-3323; Halazonetisand Kandil (1993), EMBO J., 12: 5057-5064; and Hainaut et al. (1994),Oncogene, 9: 299-303].

On the C-terminal side of the tetramerization domain (i.e., C-terminalto residue 355 of human p53), p53 contains a region that negativelyregulates DNA binding. The function of this region is abrogated bydeletion of residues 364-393 of human p53 or by deletion of the;corresponding residues of mouse p53 (residues 361-390 of the mouse p53sequence shown in[ SEQ ID NO: 3) [Hupp et al. (1992), Cell, 71: 875-886;Halazonetis et al. (1993), EMBO J., 12: 1021-1028; Halazonetis andKandil (1993), cited above].

Thus, deletion of this negative regulatory region activates DNA bindingof p53 [Halazonetis and Kandil (1993), cited above; Hupp et al. (1992),cited above]. In addition, incubation of p53 with antibody PAb421, whichrecognizes p53 at amino acids 373-381, also activates DNA binding,presumably by masking and inactivating this negative regulatory region[Hupp et al. (1992), cited above; Halazonetis et al. (1993), citedabove]. We hereinafter refer to this negative regulatory region as NRR1.(We have now developed experimental evidence which suggests that p53contains additional negative regulatory regions, see Example 3.)

Hupp et al. [(1992), cited above] have suggested that NRR1 affects theoligomerization state of wild-type p53 between tetramers and dimers. Incontrast, we had proposed that in spite of its proximity to thetetramerization domain, the NRR1 does not affect p53 oligomerization,but rather controls the conformation of p53 [Halazonetis et al. (1993),cited above]. To definitively support our model, we demonstrated thatthe activated form of p53 that lacks the NRR1 is a tetramer, as isfull-length p53 [Halazonetis and Kandil (1993), cited above]. Thus, p53switches between two conformational states, both tetrameric: an R statewith high affinity for DNA and a T state with no or low affinity for DNA[Halazonetis and Kandil (1993), cited above].

Irrespective of the mechanism by which NRR1 controls p53 DNA binding,the potential exists to develop drugs that inactivate this region andupregulate p53 DNA binding. Such drugs would be useful for treatment ofcancer, since enhanced p53 function leads to arrest of cellproliferation or to cell death [Finlay et al. (1989), Cell, 57:1083-1093; Eliyahu et al. (1985:), Proc. Natl. Acad. Sci. USA, 86:8763-8767; Baker et al. (1990), Science, 249: 912-915; Mercer et al.(1990), Proc. Natl. Acad. Sci. USA, 87: 6166-6170; Diller et al. (1990),Mol. Cell. Biol., 10: 5772-5781; Isaacs et al. (1991), Cancer Res., 51:4716-4720; Yonish-Rouach et al. (1993), Mol. Cell. Biol., 13: 1415-1423;Lowe et al. (1993), Cell, 74: 957-967; Fujiwara et al. (1993), CancerRes., 53: 4129-4133; Fujiwara et al. (1994), Cancer Res., 54:2287-2291].

Lane and Hupp have already suggested that antibody PAb421 can be usedfor the treatment of cancer, because it activates DNA binding of p53 invitro (International Patent Application WO 94/12202). However,administration of antibody PAb421 to a patient for this purpose wouldprobably be futile, since antibodies do not readily penetrate cellmembranes to reach intracellular proteins, such as p53. Lane and Huppfurther argue that any ligand, including small molecule ligands, whichbind to the C-terminal 30 amino acids of human p⁵³ would activate itsDNA binding activity (International Patent Application WO 94/12202).However, the only ligand they describe (i.e., antibody PAb421) is atleast 100 times greater in molecular size than pharmaceutical compoundsknown to penetrate cells. Their claim further lacks strength, since theC-terminal 30 amino acids of human p53 (residues 364-393 of SEQ ID NO:2) and the NRR1 do not coincide (although they do overlap).

Thus a need exists to characterize the mechanism by which NRR1 affectsDNA binding activity of p53. In particular, a need exists foridentification of small molecules which can up-regulate p53 binding ofDNA. There is a further need for methods which identify tumorsexpressing p53 mutants whose DNA binding activity can be upregulated bysmall molecules which have similar effects on wild-type p53. There isalso a need for therapeutic methods based on administering suchupregulatory molecules to cells which exhibit disease states reflectinglow p53 activity.

SUMMARY OF THE INVENTION

It is an object of this invention to provide small molecules which areable to upregulate p53 binding to DNA. Such small molecules caninterfere with the function of NRR1, but may not necessarily bind to it.

It is another object of this invention to provide small molecules whichupregulate DNA binding by p53 by interfering with the function of thenegative regulatory region (NRR1) contained within the p53 C-terminus,preferably to substantially the same extent as monoclonal antibodyPAb421.

It is a further object of this invention to provide an assay whichidentifies the small molecules within the scope of this invention and/orquantitates their activity.

It is yet a further object of this invention to provide a method foridentifying those mutant forms of p53 which exhibit DNA binding activitythat may be stimulated by the small molecules of this invention.

It is yet another object of this invention to provide a method forstimulating DNA binding activity of p53, or mutant forms of p53, in thecells of patients in need thereof, particularly in tumor cellsexpressing mutant forms of p53 having DNA binding activity which can bestimulated by the small molecules of this invention.

These and other objects are met by the invention disclosed herein.

The need set forth above for agents that activate DNA binding of humanp53 has, prompted the present inventors to further study the regulationof human p53. We have mapped NRR1 to residues 361-383 of human p53 (seeExample 3). Consistent with our mapping, antibody PAb421, which binds top53 at i.e., residues 373-381 (within the C-terminal 30 amino acids ofhuman p53) [Wade-Evans and Jenkins (1985), EMBO J., 4:699-706] activatesDNA, binding [Hupp, et al. (1992), cited above; Halazonetis, et al.(1993), cited above], whereas antibody ICA-9, which binds to p53residues 382-392 (also within the 30 C-terminal amino acids of humanp53) does not activate DNA binding [Hupp and Lane (1994), CurrentBiology 4: 865-875]. These studies have led to the identification of lowmolecular weight compounds that activate DNA binding of human p53 andare useful in the development of therapies for, and/or the preventionof, cancers, as well as other diseases and disorders caused byinadequate p53 function in vivo. These low molecular weight compoundsinclude peptide fragments and unnatural peptides whose amino acidsequence is derived from NRR1 of p53, modifications of these peptides,and peptidomimetics having the desired activity.

Thus the present invention provides peptides whose amino acid sequenceis derived from p53 and peptidomimetics based on the structure of suchpeptides, as well as methods for the use of these peptides andpeptidomimetics in therapy of cancer and other disorders characterizedby excessive proliferation of certain cells and tissues. In a preferredmode the peptides of this invention are not subfragments of p53,although their amino acid sequence is derived from the linear sequenceof human p53 or the corresponding sequences of non-human p53.Particularly, suitable non-human p53 sources include mouse, chicken,xenopus and trout.

In one aspect, the present invention provides peptides which are capableof activating the DNA binding activity of the wild-type p53 tumorsuppressor, as well as of mutant forms of p53 associated with certainhuman tumors. Such peptides include peptides which contain amino acidsequences corresponding to amino acid residues (aa) 363-373, 368-380,373-383, 371-383, 363-382, 367-386, 363-386, 362-386 and 360-386 of p53.

In another aspect, the present invention provides D-amino acid peptideswith sequences, corresponding to p53 amino acid residues (aa) 363-373,368-380, 373-383, 371-383, 363-382, 367-386, 363-386, 362-386 and360-386, but in the reverse orientation relative to human p53 (reverse-Dpeptides), which peptides are capable of activating the DNA bindingactivity of the wild-type p53 tumor suppressor, as well as of mutantforms of p53 associated with certain human tumors.

In a further aspect, the invention provides for modified versions of thepeptides described above, including both analogs that contain thepeptides described above or analogs and fragments thereof andcorresponding sequences of non-human p53 origin. All peptides of thisinvention share the ability to activate the DNA binding activity ofhuman p53.

In another aspect, the invention provides peptidomimetic compounds,which are non-peptide compounds having the same three-dimensionalstructure as the peptides of this invention or compounds in which partof a peptide according to this invention is replaced by a non-peptidemoiety having the same three-dimensional structure. The invention alsoprovides methods for selecting such peptidomimetic compounds.

Yet another aspect of this invention provides a pharmaceuticalcomposition comprising one or a combination of the above-identifiedpeptides or peptidomimetics in a pharmaceutically acceptable carrier.

Still other aspects of this invention involve methods of using thepharmaceutical compositions of this invention for activating p53function in human subjects. Such activation would: 1) induce thecellular response to DNA damaging agents, thereby increasing theresistance of healthy subjects to DNA damaging agents, such as sunlight,radiation, etc., and reducing the toxicity of therapies employing DNAdamaging agents, such as cancer therapy, 2) induce apoptosis oflymphocytes, thereby conferring immune tolerance for patients withautoimmune diseases, allergies or for transplant recipients, 3) enhancep53 function of abnormally proliferating cells, such as those associatedwith cancer, psoriasis, etc., thereby leading to treatment by apoptosisor growth arrest of such cells.

Without wishing to be bound by theory, the inventors selected thepeptides of this invention from the complete p53 sequence based on theirfinding that two negative regulatory regions exist in p53, i.e., thesequence from residues 300-321 of human p53 (NRR2) and the previouslyrecognized regulatory region approximately within residues 361-383 ofhuman p53 (NRR1). The inventors hypothesized that these two sequencesphysically interact with each other or with a third region in p53,shifting p53 into a conformation with low affinity for DNA. When theinteractions of these two negative regulatory regions are disrupted (bydeletions within one of the two regions or by a suitable antibody), thenp53 shifts into a conformation with high affinity for DNA.

By competing with the endogenous p53 negative regulatory region forbinding to these regions, small molecules may disrupt the intramolecularregulatory interactions of p53. The inventors designed peptidescorresponding to one of these negative regulatory regions, and observedthat the peptides of this invention activate DNA binding of human p53.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

According to this invention, peptides are provided that activate the DNAbinding activity of the wild-type form of the p53 tumor suppressor, aswell as of certain tumor-derived p53 mutants. The mutants which can beso activated are among the most significant for human tumors. Mutantswhich may be activated by the peptides of this invention include thosecharacterized by a single amino acid residue modification (substitution)at the following locations in the sequence of p53: (a) Ser at residue239; (b) His at residue 273; (c) Gln at residue 248; (d) Trp at residue282; and (e) Cys at residue 273. On the other hand, peptides accordingto this invention do not activate p53 mutants having only substitutionof: His at position 175, Trp at position 248, Ser at position 249 andIle at position 237.

The small molecules of this invention include peptide derivatives whichretain the effective sequence of peptides and are effective in a p53 DNAbinding assay (as measured, for example, by the assay procedure inExample 2). The present invention provides small molecule's that arepeptides which contain amino acid sequences from NRR1 of p53, modifiedforms of the peptides (which retain the activity of the peptides), orpeptidomimetics (which retain the essential three-dimensional shape andchemical reactivity, and therefore the biological activity, of thepeptides). The small molecules of this invention usually have molecularweight less than 2,000 daltons (Da), preferably less than 1,500 Da, morepreferably less than 1,000 Da, most preferably less than 500 Da, andthese small molecules activate DNA binding of wild-type p53 atconcentrations of 0.2 mM or lower with the same efficacy as peptidesmade up of p53 residues 363-373, 368-380, 373-383, 371-383, 363-382,367-386, 363-386, 362-386 or 360-386.

I. PEPTIDES OF THE INVENTION

Peptides of this invention contain amino acid sequences corresponding toat least a part of NRR1 of p53 (residues 361-383 of human p53). Thesepeptides include the following:

peptide p53p363-373 [SEQ ID NO: 4] Arg Ala His Ser Ser His Leu Lys SerLys Lys peptide p53p368-380 [SEQ ID NO: 5] His Leu Lys Ser Lys Lys GlyGln Ser Thr Ser Arg His peptide p53p373-383 [SEQ ID NO: 6] Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu peptide p53p371-383 [SEQ ID NO: 7] SerLys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu peptide p53p363-382 [SEQID NO: 8] Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser ThrSer Arg His Lys Lys peptide p53p367-386 [SEQ ID NO: 9] Ser His Leu LysSer Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys peptidep53p363-386 [SEQ ID NO: 10] Arg Ala His Ser Ser His Leu Lys Ser Lys LysGly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys peptide p53p362-386[SEQ ID NO: 11] Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu Met Phe Lys peptide p53p360-386 [SEQ IDNO: 12] Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu Met Phe Lys peptide p53p363-370 [SEQ IDNO: 14] Arg Ala His Ser Ser His Leu Lys peptide p53p368-373 [SEQ ID NO:15] His Leu Lys Ser Lys Lys peptide p53-368-372 [SEQ ID NO: 16] His LeuLys Ser Lys peptide p53p369-373 [SEQ ID NO: 17] Leu Lys Ser Lys Lyspeptide p53p370-375 [SEQ ID NO: 18] Lys Ser Lys Lys Gly Gln peptidep53p370-374 [SEQ ID NO: 19] Lys Ser Lys Lys Gly

It will be apparent to one of skill in the art that other smallerfragments of the above peptides may be selected by one of skill in theart and these peptides will possess the same biological activity. As anexample, fragments of the peptide p53p360-386 [SEQ ID NO: 12], rangingin length from 26 amino acids to about 5 amino acids, are includedwithin this invention. In general, the peptides of this invention haveat least 4 amino acids, preferably at least 5 amino acids, morepreferably at least 6 amino acids.

The peptides of this invention also include peptides having sequences ofnon-human p53 segments corresponding to the NRR1 region. The amino acidsequence of p53 is conserved across species [Soussi et al. (1990),Oncogene, 5: 945-952 incorporated herein by reference], implying thatfunction is also conserved. Indeed, analysis of xenopus and human p53proteins has revealed no functional differences [Cox et al. (1994),Oncogene, 9: 2951-2959]. Thus, it is possible to substitute human p53sequences of the peptides of this invention with the homologousnon-human p53 sequences. The sequences of human p53 and select non-humanp53 proteins have been aligned by Soussi et al. (1990) [cited above].This alignment can serve lo identify regions that are homologous acrossspecies. For p53 species that are not listed by Soussi et al. (1990)[cited above], the alignment to the human p53 sequences can be obtainedby computer programs commercially available and known in the art, suchas the program BESTFIT of the University of Wisconsin GCG package. Theentire peptide sequences presented above can be substituted by thecorresponding non-human sequences, or alternatively, a fragment of theabove peptide sequences can be substituted by the correspondingnon-human sequences.

While the peptides described above are effective in activating DNAbinding of wild-type p53 in vitro, their effectiveness in vivo might becompromised by the presence of proteases. Serum proteases have quitespecific substrate requirements. The substrate must have both L-aminoacids and peptide bonds for cleavage. Furthermore, exopeptidases, whichrepresent the most prominent component of the protease activity inserum, usually act on the first peptide bond of the peptide and requirea free N-terminus (Power, et al. (1993), Pharmaceutical Res.,10:1268-1273). Based on these considerations, it is advantageous toutilize modified versions of the peptides described above. The modifiedpeptides retain the structural characteristics of the original L-aminoacid peptides that confer biological activity with regard to p53, butbecause of the modification, they are not readily susceptible tocleavage by proteases and/or exopeptidases.

As contemplated by this invention, the term “peptide” includes modifiedforms of the peptide, so long as the modification does not alter theessential sequence and the modified peptide retains the ability toactivate p53 binding to specific DNA sequences (i.e., sequence specificDNA binding activity). Such modifications include N-terminalacetylation, glycosylation, biotinylation, etc. Particular modifiedversions of the L-amino acid peptides corresponding to the amino acidsequence of the p53 NRR1 (residues 361-383 of human p53) are describedbelow and are considered to be peptides according to this invention:

A. Peptides With An N-Terminal D-Amino Acid

The presence of an N-terminal D-amino acid increases the serum stabilityof a peptide which otherwise contains L-amino acids, becauseexopeptidases acting on the N-terminal residue cannot utilize a D-aminoacid as a substrate (Powell, et al. (1993), cited above). Thus, theamino acid sequences of the peptides with N-terminal D-amino acids areusually identical to the sequences of the L-amino acid peptidesdescribed above [e.g., SEQ ID NO: 4-12], except that the N-terminalresidue is a D-amino acid.

B. Peptides With A C-Terminal D-Amino Acid

The presence of an C-terminal D-amino acid also stabilizes a peptide,which otherwise contains L-amino acids, because serum exopeptidasesacting on the C-terminal residue cannot utilize a D-amino acid as asubstrate (Powell, et al. (1993), cited above). Thus, the amino acidsequences of the these peptides are usually identical to the sequencesof the L-amino acid peptides described above [e.g., SEQ ID NO: 4-12],except that the C-terminal residue is a D-amino acid.

C. Cyclic Peptides

Cyclic peptides have no free N- or C-termini. Thus, they are notsusceptible to proteolysis by exopeptidases, although they are of coursesusceptible to endopeptidases, which do not cleave at peptide termini.The amino acid sequences of the cyclic peptides may be identical to thesequences of the L-amino acid peptides described above [e.g., SEQ ID NO:4-12], except that the topology is circular, rather than linear.

D. Peptides With Substitution of Natural Amino Acids By Unnatural AminoAcids

Substitution of unnatural amino acids for natural amino acids in asubsequence of the NRR1 of p53 can also confer resistance toproteolysis. Such a substitution can, for example, confer resistance toproteolysis by exopeptidases acting on the N-terminus. Several of thepeptides whose amino acid sequence is derived from the NRR1 of human p53(residues 361-383 of human p53) have serine as the N-terminal residue(see for example SEQ ID NO: 7, 9, and 11). The serine residue can besubstituted by the 0-amino acid isoserine. Such substitutions have beendescribed (Coller, et al. (1993), J. Biol. Chem., 268:20741-20743,incorporated herein by reference) and these substitutions do not affectbiological activity. Furthermore, the synthesis of peptides withunnatural amino acids is routine and known in the art (see, for example,Coller, et al. (1993), cited above).

E. Peptides With N-Terminal or C-Terminal Chemical Groups

An effective approach to confer resistance to peptidases acting on theN-terminal or C-terminal residues of a peptide is to add chemical groupsat the peptide termini, such that the modified peptide is no longer asubstrate for the peptidase. One such chemical modification isglycosylation of the peptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of peptides in human serum [Powellet al. (1993), Pharma. Res., 10: 1268-1273]. Other chemicalmodifications which enhance serum stability include, but are not limitedto, the addition of an N-terminal alkyl group, consisting of a loweralkyl of from 1 to 20 carbons, such as an acetyl group, and/or theaddition of a C-terminal amide or substituted amide group. In particularthe present invention includes modified peptides consisting of residues363-373, 368-380, 373-383, 371-383, 363-382, 367-386, 363-386, 362-386or 360-386 of human p53 bearing an N-terminal acetyl group and aC-terminal amide group.

F. Peptides With Additional Amino Acids

Also included within this invention are modified peptides which containwithin their sequences the peptides described above. These longerpeptide sequences, which result from the: addition of extra amino acidresidues are encompassed in this invention, since they have the samebiological activity (i.e., activate DNA binding of p53) as the peptidesdescribed above.

One specific example of variants of the peptide corresponding to aminoacids residues 360-386 of human p53 includes the addition of anN-terminal cysteine which confers to the peptide the ability to formdimers:

peptide p53pC360-386 [SEQ ID NO: 22] Cys Gly Gly Ser Arg Ala His Ser SerHis Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met PheLys

While peptides having a substantial number of additional amino acids arenot excluded, it will be recognized that some large polypeptides willassume a configuration that masks the effective sequence, therebypreventing binding to p53. Other polypeptides will still bind, but areso bulky that the complex of p53 with peptides will no longer bind toDNA. These derivatives will not enhance p53 action and are therebyexcluded from the invention.

G. Peptides With Deleted Amino Acids

Peptides of this invention have amino acid sequences contained withinNRR1 of p53 To the extent that a peptide containing the sequence of asegment of NRR1 has the desired biological activity, it follows that apeptide that contains the sequences of two such segments, would alsopossess the desired biological activity, even if these segments were notcontiguous within the p53 NRR1. Such peptides can also be described ashaving a sequence corresponding to the p53 NRR1 with an internaldeletion.

The ability of peptides with internal deletions to activate DNA bindingof p53 was first realized by the observation that synthesis of peptidep53pC360-386 [SEQ ID NO: 22] also generated peptides lacking a glycineor phenylalanine, or combinations thereof, such as:

peptide p53pC360-386DG [SEQ ID NO: 23] Cys Gly Ser Arg Ala His Ser SerHis Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met PheLys peptide p53pC360-386DF [SEQ ID NO: 24] Cys Gly Gly Ser Arg Ala HisSer Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys LeuMet Lys peptide p53pC360-386DGF [SEQ ID NO: 25] Cys Gly Ser Arg Ala HisSer Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys LeuMet Lys

The ability of the above peptides to activate DNA binding of p53 hasprompted us to design additional peptides with single amino aciddeletions or longer deletions.

Modified peptides which contain single amino acid deletions alsoinclude:

peptide p53p363-370D366 [SEQ ID NO: 20] Arg Ala His Ser His Leu Lyspeptide p53p368-373D369 [SEQ ID NO: 21] His Lys Ser Lys Lys peptidep53p370-375D374 [SEQ ID NO: 32] Lys Ser Lys Lys Gln

Modified peptides according to this invention, having longer deletions,include:

peptide p53p363-373D369-371 [SEQ ID NO: 33] Arg Ala His Ser Ser His LysLys peptide p53p368-380D372-378 [SEQ ID NO: 34] His Leu Lys Ser Arg His

H. Reverse-D Peptides

In another embodiment of this invention the peptides are reverse-Dpeptides corresponding to the amino acid sequence of the p53 NRR1(residues 361-386 of human p5:.). The term “reverse-D peptide” refers topeptides containing D-amino acids, arranged in a reverse sequencerelative to a peptide containing L-amino acids. Thus, the C-terminalresidue of an L-amino acid peptide becomes N-terminal for the D-aminoacid peptide, and so forth. For example, the sequence of the reverse-Dpeptide corresponding to peptide p53p363-373 [SEQ ]D NO: 4] is:

peptide p53RDp363-373 [SEQ ID NO: 13]Lys Lys Ser Lys Leu His Ser Ser HisAla Arg

The reverse-D sequences of the peptides with SEQ ID NOS: 5-11 arepresented as SEQ ][D NOS: 13-21. Reverse-D peptides retain the sametertiary conformation, and therefore the same activity, as the L-aminoacid peptides, but are more stable to enzymatic degradation in vitro andin vivo, and thus have greater therapeutic efficacy than the originalpeptide (Brady and Dodson (1994), Nature, 368: 692-693; Jameson et al.(1994), Nature, 368: 744-746).

The peptides of this invention, including the analogs and other modifiedvariants, may generally be prepared following known techniques.Preferably, synthetic production of the peptide of the invention may beaccording to the solid phase synthetic method. For example, the solidphase synthesis is well understood and is a common method forpreparation of peptides, as are a variety of modifications of thattechnique [Merrifield (1964), J. Am. Chem. Soc., 85: 2149; Stewart andYoung (1984), Solid Phase Peptide Synthesis, Pierce Chemical Company,Rockford, Ill.; Bodansky and Bodanszky (1984), The Practice of PeptideSynthesis, Springer-Verlag, New York; Atherton and Sheppard (1989),Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, NewYork]. See, also, the specific method described in Example 1 below.

Alternatively, peptides of this invention may be prepared in recombinantsystems using polynucleotide sequences encoding the peptides. It isunderstood that a peptide of this invention may contain more than one ofthe above described modifications within the same peptide. Also includedin this invention are pharmaceutically acceptable salt complexes of thepeptides of this invention.

II. PEPTIDOMIMETICS

A peptide mimetic is a molecule that mimics the biological activity of apeptide but is no longer peptidic in chemical nature. By strictdefinition, a peptidomimetic is a molecule that no longer contains anypeptide bonds (that is, amide bonds between amino acids). However, theterm peptide mimetic is sometimes used to describe molecules that are nolonger completely peptidic in nature, such as pseudo-peptides,semi-peptides and peptoids. Examples of some peptidomimetics by thebroader definition (where p of a peptide is replaced by a structurelacking peptide bonds) are described below. Whether completely orpartially non-peptide, peptidomimetics according to this inventionprovide a spatial arrangement of reactive chemical moieties that closelyresembles the three-dimensional arrangement of active groups in thepeptide on which the peptidomimetic is based. As a result of thissimilar active-site geometry, the peptidomimetic has effects onbiological systems which are similar to the biological activity of thepeptide.

The present invention encompasses peptidomimetic compositions which areanalogs that mimic the activity of biologically active peptidesaccording to the invention, i.e., the peptidomimetics are capable ofactivating the DNA binding activity of p53. The peptidomimetic of thisinvention are preferably substantially similar in both three-dimensionalshape and biological activity to the peptides set forth above.Substantial similarity means that the geometric relationship of groupsin the peptide that react with p53 is preserved and at the same time,that the peptidomimetic will stimulate the DNA binding activity ofwild-type p53 and one or more of the p53 mutants set forth above, thestimulation being within a factor of two of the stimulation exhibited byat least one of the peptides of this invention.

There are clear advantages for using a mimetic of a given peptide ratherthan the peptide itself, because peptides commonly exhibit twoundesirable properties: (1) poor bioavailability; and (2) short durationof action. Peptide mimetics offer an obvious route around these twomajor obstacles, since the molecules concerned are small enough to beboth orally active and have a long duration of action. There are alsoconsiderable cost savings and improved patient compliance associatedwith peptide mimetics, since they can be administered orally comparedwith parenteral administration for peptides. Furthermore, peptidemimetics are much cheaper to produce than peptides. Finally, there areproblems associated with stability, storage and immunoreactivity forpeptides that are not experienced with peptide mimetics.

Thus peptides described above have utility in the development of suchsmall chemical compounds with similar biological activities andtherefore with similar therapeutic utilities. The techniques ofdeveloping peptidomimetics are conventional. Thus, peptide bonds can bereplaced by non-peptide bonds that allow the peptidomimetic to adopt asimilar structure, and therefore biological activity, to the originalpeptide. Further modifications can also be made by replacing chemicalgroups of the amino acids with other chemical groups of similarstructure. The development of peptidomimetics can be aided bydetermining the tertiary structure of the original peptide, either freeor bound to p53, by NMR spectroscopy, crystallography and/orcomputer-aided molecular modelling. These techniques aid in thedevelopment of novel compositions of higher potency and/or greaterbioavailability and/or greater stability than the original peptide [Dean(1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J. Mol.Graph., 11: 166-173; Wiley and Rich (1993), Med. Res. Rev., 13: 327-384;Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby (1993),Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98,all incorporated herein by reference]. Once a potential peptidomimeticcompound is identified, it may be synthesized and assayed using the DNAbinding assay described herein or an appropriate tumor suppressor assay[see, Finlay et al. (1983), Cell, 57: 1083-1093 and Fujiwara et al.(1993), Cancer Res., 53: 4129-4133, both incorporated herein byreference], to assess its activity.

Thus, through use of the methods described above, the present inventionprovides compounds exhibiting enhanced therapeutic activity incomparison to the peptides described above. The peptidomimetic compoundsobtained by the above methods, having the biological activity of theabove named peptides and similar three dimensional structure, areencompassed by this invention. It will be readily apparent to oneskilled in the art that a peptidomimetic can be generated from any ofthe modified peptides described in the previous section or from apeptide bearing more than one of the modifications described from theprevious section. It will furthermore be apparent that thepeptidomimetics of this invention can be further used for thedevelopment of even more potent non-peptidic compounds, in addition totheir utility as therapeutic compounds.

Specific examples of peptidomimetics derived from the peptides describedin the previous section are presented below. These examples areillustrative and not limiting in terms of the other or additionalmodifications.

A. Peptides With A Reduced Isostere Pseudopeptide Bond [↓(CH₂NH)]

Proteases act on peptide bonds. It therefore follows that substitutionof peptide bonds by pseudopeptide bonds confers resistance toproteolysis. A number of pseudopeptide bonds have been described that ingeneral do not affect peptide structure and biological activity. Thereduced isostere pseudopeptide bond is a suitable pseudopeptide bondthat is known to enhance stability to enzymatic cleavage with no orlittle loss of biological activity (Couder, et al. (1993), Int. J.Peptide Protein Res., 41:181-184, incorporated herein by reference).Thus, the amino acid sequences of these peptides may be identical to thesequences of the L-amino acid peptides described above [e.g., SEQ ID NO:4-12], except that one or more of the peptide bonds are replaced by anisostere pseudopeptide bond. Preferably the most N-terminal peptide bondis substituted, since such a substitution would confer resistance toproteolysis by exopeptidases acting on the N-terminus. The synthesis ofpeptides with one or more reduced isostere pseudopeptide bonds is knownin the art (Couder, et al. (1993), cited above).

B. Peptides With A Retro-Inverso Pseudopeptide Bond [↓(NHCO)]

To confer resistance to proteolysis, peptide bonds may also besubstituted by retro-inverso pseudopeptide bonds (Dalpozzo, et al.(1993), Int. J. Peptide Protein Res., 41:561-566, incorporated herein byreference). According to this modification, the amino acid sequences ofthe peptides may be identical to the sequences of the L-amino acidpeptides described above [e.g., SEQ ID NO: 4-12], except that one ormore of the peptide bonds are replaced by a retro-inverso pseudopeptidebond. Preferably the most N-terminal peptide bond is substituted, sincesuch a substitution will confer resistance to proteolysis byexopeptidases acting on the N-terminus. The synthesis of peptides withone or more reduced retro-inverso pseudopeptide bonds is known in theart (Dalpozzo, et al. (1993), cited above).

C. Peptoid Derivatives

Peptoid derivatives of peptides represent another form of modifiedpeptides that retain the important structural determinants forbiological activity, yet eliminate the peptide bonds, thereby conferringresistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci.US., 89:9367-9371 and incorporated herein by reference). Peptoids areoligomers of N-substituted glycines. A number of N-alkyl groups havebeen described, each corresponding to the side chain of a natural aminoacid (Simon, et al. (1992), cited above and incorporated herein byreference). Thus, the sequence of N-alkyl groups of a peptoidcorresponding to peptide p53p363-373 [SEQ ID NO: 4] would be:

Narg Nala Nhhis Nhser Nhser Nhhis Nleu Naeg Nhser Naeg Naeg

where the correspondence of N-alkyl groups of the peptoid to the naturalamino acids is: Narg→Arg Nala→Ala Nhhis→His Nhser→Ser Naeg→Lys. Thedesignation of the peptoid N-alkyl groups follows Simon, et al. (1992)(cited above).

While the example indicated above replaces every amino acid of thepeptide with the corresponding N-substituted glycine, it is obvious thatnot all of the amino acids have to be replaced. For example theN-terminal residue may be the only one that is replaced, or a few aminoacids may be replaced by the corresponding N-substituted glycines.

III. PHARMACEUTICAL COMPOSITIONS

The ability of the above-described peptides and compositions of thisinvention to activate the DNA binding activity of p53 and thus activatethe cellular functions of p53 described above, enables their use aspharmaceutical compositions in a variety of therapeutic regimens. Thepresent invention therefore includes novel therapeutic pharmaceuticalcompositions and methods for treating a human or animal with suchcompositions. As used herein, the term “pharmaceutical” includesveterinary applications of the invention.

To prepare the pharmaceutical compositions of the present invention, atleast one peptide (or peptidomimetic), or alternatively, a mixture ofpeptides (or peptidomimetics) of this invention is combined as theactive ingredient in intimate admixture with a pharmaceutical carrierselected and prepared according to conventional pharmaceuticalcompounding techniques. This carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral, sublingual, rectal, nasal, or parenteral.

Pharmaceutically acceptable solid or liquid carriers or components whichmay be added to enhance or stabilize the composition, or to facilitatepreparation of the composition include, without limitation, syrup,water, isotonic saline solution, 5% dextrose in water or buffered sodiumor ammonium acetate solution, oils, glycerin, alcohols, flavoringagents, preservatives, coloring agents starches, sugars, diluents,granulating agents, lubricants, and binders, among others. The carriermay also include a sustained release material such as glycerylmonostearate or glyceryl distearate, alone or with a wax. The amount ofsolid carrier varies but, preferably will be between about 20 mg toabout 1 g per dosage unit.

Pharmaceutical compositions of the peptides of this invention, orderivatives thereof, may therefore be formulated as solutions oflyophilized powders for parenteral administration. The presentlypreferred method is that of intravenous administration.

Pharmaceutical compositions of this invention may also include topicalformulations incorporated in a suitable base or vehicle, for applicationat the site of the area for the exertion of local action. Accordingly,such topical compositions include those forms in which the formulationis applied externally by direct contact with the skin surface to betreated.

Conventional forms for this purpose include but are not limited tocreams, ointments, lotions, gels, pastes, powders and formulationshaving oleaginous absorption, water-soluble, and emulsion-type bases.

Additionally, the compounds of the present invention may also beadministered encapsulated in liposomes. The compositions, depending uponits solubility, may be present both in the aqueous layer and in thelipidic layer, or in what is generally termed a liposomic suspension.The hydrophobic layer, generally but not exclusively, comprisesphospholipids such as lecithin and sphingomyelin, steroids such ascholesterol, more or less ionic surfactants such a diacetylphosphate,stearylamine, or phosphatidic acid, and/or other materials of ahydrophobic nature.

The compositions may be supplemented by active pharmaceuticalingredients, where desired. Optional antibacterial, antiseptic, andantioxidant agents may also be present in the compositions where theywill perform their ordinary functions.

Dosage units of such pharmaceutical compositions containing the peptidesor peptidomimetic compounds of this invention preferably contain about 1mg-5 g of the peptide or salt thereof.

As used herein, the terms “suitable amounts” or “therapeuticallyeffective amount” means an amount which is effective to treat theconditions referred to below. A peptide or peptidomimetic of the presentinvention is generally effective when parenterally administered inamounts above about 1 mg per kg of body weight to about 30 mg/kg.

IV. METHODS OF TREATMENT/UTILITIES

The pharmaceutical compositions described above and identified with theability to activate the DNA binding activity of p53 are useful intherapeutic regimens which exploit the cellular functions of p53.

As one example, the pharmaceutical compositions of this invention may beemployed to induce the cellular response to DNA damaging agents, such asUV irradiation, radiation and chemotherapeutics used for cancertreatment. By administering a suitable amount of a composition of thisinvention, patients may tolerate higher doses of such DNA damagingagents. For example, pharmaceutical compositions of this invention maytake the form of sunscreens or other sun protective compositions whichare administered topically. Alternatively, compositions of thisinvention may be administered parenterally (for example, intravenously)as an adjunct to patients receiving traditional cancer therapy, whichemploys the use of DNA damaging agents (eg. radiation therapy andchemotherapy). Other modes of administration may be employed whereappropriate.

The compositions of this invention may also be employed as the soletreatment for patients with cancer to enhance the tumor suppressorfunction of p53, whether wild-type or mutant, present in tumor cells.The administration of the composition to a cancer patient thus permitsthe arrest of the growth or proliferation of tumor cells or apoptosis(cell death) of tumor cells. Desirably, a suitable amount of thecomposition of this invention is administered systemically, or locallyto the site of the tumor.

Additionally, the compositions of this invention may be administered inmethods to suppress cell proliferation in diseases other than cancers,which are characterized by aberrant cell proliferation. Among suchdiseases are included psoriasis, atherosclerosis and arterialrestenosis. This method is conducted by administering a suitable amountof the selected composition topically, locally or systemically to apatient.

Another therapeutic use of the compositions of this invention is ininducing apoptosis of specific cells, such as proliferating lymphocytes.According to this method of use, a suitable amount of a composition ofthis invention is administered to a subject to enhance the developmentof immune tolerance. This method may employ both in vivo and ex vivomodes of administration. Preferably, this therapy is useful as the soletreatment or as an accessory treatment to prevent transplant rejectionor to treat autoimmune diseases, e.g., systemic lupus; erythrematosis,rheumatoid arthritis and the like.

The peptides and peptidomimetics of the invention may also be utilizedin methods for monitoring disease progression, particularly in a patientreceiving therapy as provided by the present invention, or to determinewhich patients are suited for this therapy. Such a method involvesobtaining a tumor biopsy from the patient, preparing an extract[Halazonetis et al. (1993), cited above], and testing this extract forp53-dependent DNA binding in the presence and absence of a peptide orpeptidomimetic of the invention (e.g., as described in Example 2,below). If the peptide increases DNA binding, then therapeutic use ofthe compositions of this invention is indicated and outcome of therapyis improved.

EXAMPLES

The following examples illustrate the preferred compositions and methodsof this invention. In view of this disclosure, it will be clear to oneof skill in the art that other useful fragments and analogs of thepeptides described herein are readily identifiable by one of skill inthe art, and are therefore encompassed in this invention. These examplesare illustrative only and do not limit the scope of the invention.

Example 1

Synthesis of Peptides

Peptides corresponding to the negative regulatory region 1 (NRR1)spanning residues 361-383 of human p53 were synthesized. Peptides wereassembled on a Milligen 9050 automated synthesizer using the standardFmoc-protocol [Fields and Noble (1990), Int. J. Pept. Protein Res., 35:161-214]. After cleavage, the peptides were purified by reverse phaseHPLC using a C18 column and a linear gradient of acetonitrile in 0.1%aqueous trifluoroacetic acid. The efficiency and accuracy of synthesiswas monitored by amino acid composition analysis and/or massspectroscopy. The present invention is not limited by the specificchemistry or synthesizer used to prepare the peptides of this invention;and such factors are not critical for the activities of the peptides ofthis invention.

The following peptides were synthesized:

peptide p53p363-373 [SEQ ID NO: 4] Arg Ala His Ser Ser His Leu Lys SerLys Lys peptide p53p368-380 [SEQ ID NO: 5] His Leu Lys Ser Lys Lys GlyGln Ser Thr Ser Arg His peptide p53p373-3B3 [SEQ ID NO: 6] Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu peptide p53p371-3B3 [SEQ ID NO: 7] SerLys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu peptide p53p363-3B2 [SEQID NO: 8] Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser ThrSer Arg His Lys Lys peptide p53p367-386 [SEQ ID NO: 9] Ser His Leu LysSer Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys peptidep53p363-386 [SEQ ID NO: 10] Arg Ala His Ser Ser His Leu Lys Ser Lys LysGly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys peptide p53p362-386[SEQ ID NO: 11] Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu Met Phe Lys peptide p53p360-386 [SEQ IDNO: 12] Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu Met Phe Lys peptide p53pC360-3B6 [SEQ IDNO: 22] Cys Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys GlyGln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys

All peptides were acetylated at their N-terminus and contained an amidechemical group attached to their C-terminus.

Amino acid composition analysis and mass spectroscopy indicated thatpeptide p53pC360-386 actually consisted of a mixture of peptides. Inaddition to the expected peptide, the mixture contained peptides lackinga glycine, a phenylalanine or combinations thereof [SEQ ID NOS: 23, 24and 25]. Because the latter peptides differ from the expected peptideonly at their termini, all these peptides are expected to bebiologically active. This is supported by the demonstration below thatvarious peptides corresponding to the central region of p53pC360-386have biological activity.

Each peptide was dissolved in a conventional buffer, e.g., 10 or 100 mMTris-Cl buffer pH 8 at a concentration of 10 mg/ml. Any buffer in whichthe peptides are soluble, and which is compatible with the DNA bindingassays described below, may be used. Once dissolved, the peptides werestored at −70° C.

Example 2

DNA Binding Assay

The ability of the peptides of this invention to activate DNA binding ofhuman p53 was assayed using a standard DNA binding assay for p53[Halazonetis et al. (1993), cited above; Halazonetis and Kandil (1993),cited above]. This assay utilizes in vitro translated human wild-typep53 or tumor-derived p53 mutants and specific oligonucleotidescontaining p53 binding sites. These reagents and methods for preparingthem are described below.

Human p53 was produced by in vitro translation using plasmids containingthe full-length p53 coding sequence. Standard cloning procedures[Ausubel et al. (1994), “Current Protocols in Molecular Biology,” GreenePublishing Associates and John Wiley & Sons, New York; Innis et al.(1990), PCR Protocols: A Guide to Methods and Applications, AcademicPress, San Diego] were used to prepare these plasmids.

Reagents

Plasmid pGEMhump53wt encodes full-length human wild-type p53. Thisplasmid was prepared by PCR [Innis et al. (1990), cited above] using ahuman p53 cDNA, which is readily available to those practicing the art.The PCR procedure was designed to incorporate unique restriction siteswithin human p53: Kpn I at codon 218, Sst I at codon 299, Sst II atcodon 333, Bst BI at codon 338 and Sal I immediately following thetermination codon. An Msc I site: at codon 138 was eliminated. Thesechanges did not alter the amino acid sequence of the encoded p53, andwere only performed to expedite construction of mutant proteins bearingpoint mutations associated with human cancer. The PCR product of thehuman p53 cDNA was digested with Nco I and Sal I and cloned in thevector pGEM4 [Promega, Madison, Wis.], which was linearized with Eco RIand Sal I. Synthetic oligonucleotides were used to bridge the Eco RIsite of the vector and the Nco I site at the initiation codon of p53.The entire nucleotide sequence of the Eco RI-Sal I human p53 insert inplasmid pGEMhump53wt is presented as SEQ ID NO: 26. Plasmid pGEMhump53wtwas used to generate all the p53 mutants described below, as well as forexpression of wild-type p53 by in vitro translation.

Plasmid pGEMhump53wt was used to generate plasmids encoding mutant p53proteins frequently associated with human cancer. The mutationsintroduced are those that are most frequently associated with humantumors [Caron de Fromentel et al. (1992), Genes Chrom. Cancer, 4: 1-15].Specifically the following mutants (all single amino acid substitutionmutants) were generated: His175, has histidine at position 175 of p53;Gln248, has glutamine at positions 248 of p53; Trp248, has tryptophan atposition 248 of p53; Ser249, has serine at position 245 of p53; His273,has histidine at position 273 of p53; Cys273, has cysteine at position273 of p53; Trp282, has tryptophan at position 282 of p53; Ser239, hasserine at position 239 of p53; and Ile237, has isoleucine at position237 of p53. All p53 mutants were generated using PCR [Innis et al.(1990), cited above]. DNA fragments containing the desired mutationswere cloned into pGEMhump53wt using the most convenient restrictionsites and confirmed by sequencing. The resultant plasmids are namedpGEMhump53His175, etc.

Plasmids pGEMhump53D300-308, pGEMhump53D300-317, pGEMhump53D300-321,pGEMhump53D356-393, pGEMhump53D364-393, pGEMhump53D379-393,pGEMhump53D384-393 and pGEMhump53D388-393 encode proteins that containdeletions within human wild-type p53. These deletions involve residues300-308, 300-317, 300-321, 356-393, 364-393, 379-393, 384-393 and388-393 of p53 SEQ ID NO: 2, respectively. These deletions weregenerated using standard recombinant techniques [Ausubel et al. (1994),cited above; Innis et al. (1990), cited above].

Assay Procedure

Plasmids of the pGEMhump53 series were used to produce in vitrotranscribed mRNA and subsequently in vitro translated protein, accordingto standard procedures [Halazonetis et al. (1988), Cell, 55: 917-924].The in vitro translated proteins were assayed for DNA binding, aspreviously described [Halazonetis et al. (1993), cited above, andincorporated herein by reference). Briefly the in vitro translatedprotein is incubated with a radioactively labeled oligonucleotidecontaining a p53 binding site in the presence of non-specific competitorDNA. The reaction mixture is incubated 20 min. at room temperature anddirectly loaded on a native 5% polyacrylamide electrophoresis gel. Inthis type of DNA binding assay free DNA migrates to the bottom of thegel, whereas p53/DNA complexes migrate more slowly. Thus, the presenceof slowly migrating DNA, which can be detected by autoradiography,indicates p53 DNA. binding [Halazonetis et al. (1993), cited above;Halazonetis and Kandil (1993), cited above].

As non-specific competitor DNAs we used 0.1 μg single-strandedoligonucleotide M17 [GAGAGCCCCAGTTACCATAACTACTCT, SEQ ID NO: 27] and0.05 μg double-stranded oligonucleotide TF3[ATCACGTGATATCACGTGATATCACGTGAT, SEQ ID NO: 28] per reaction.

A number of double-stranded oligonucleotides containing p53 bindingsites were radioactively labeled for these experiments. These includedoligonucleotides Ewaf1, BC.V4A and BC. Oligonucleotides BC.V4A and BCcontain artificial sites recognized by p53. These three sites areindicated below, and the specific pentanucleotide repeats recognized byp53 are demarcated by hyphens. The sequence of oligonucleotide Ewaf1(top strand) is: CCC-GAACA-TGTCC-CAACA-TGTTG-GGG [SEQ ID NO: 29]. Thisoligonucleotide corresponds to the enhancer that drives p53-dependenttranscription of the waf1 gene [El-Deiry et al. (1993), Cell, 75:817-825]. The sequence of oligonucleotide BC.V4A (top strand) is:TC-GAGCA-TGTTC-GAGCA-TGTTC-GAGCATGT [SEQ ID NO: 30], and the sequence ofoligonucleotide BC (top strand) is: CC-GGGCA-TGTCC-GGGCA-TGTCC-GGGCATGT[SEQ ID NO: 31]. These DNAs were radioactively labeled using 32P-labelednucleotides [Halazonetis et al. (1988), cited above].

Results using this assay are presented in Examples 3 through 5, below.

Example 3

Mapping of Negative Regulatory Regions Within Wild-type p53

The p53 DNA binding assay involves incubating in vitro translated p53with radioactively-labeled DNAs containing p53 binding sites asdescribed in Example 2 above. For these studies, incubation wasperformed with oligonucleotides BC.V4A. Wild-type p53 binds to thisoligonucleotide. However, its DNA binding activity is subject todownregulation by the negative regulatory region 1 (NRR1), which maps tothe C-terminus of p53. Thus deletion of residues 364-393 of human p53activates DNA binding [Hupp et al. (1992), cited above; Halazonetis andKandil (1993), cited above]. We reproduced this result usingoligonucleotide BC.V4A and plasmid pGEMhump53D364-393. However, DNAbinding of a deletion mutant lacking residues 353-360 was not activated(see Table 1).

To map more finely the NRR1 and to identify other NRR that might bepresent in the C-terminus of p53, we examined the DNA binding activitiesof the p53 deletion mutants described in Example 2 above. As shown inTable 1, mutants p53D356-393, p53D364-393, p53D379-393 were activatedfor binding to oligonucleotide BC.V4A. In contrast mutants p53D384-393and p53D388-393 were not. These results map the C-terminal boundary ofthe NRR1 to residue 383 of p53. The N-terminal boundary of NRR1 iscertainly C-terminal to residue 355 of p53, since residues 322-355 ofp53 form the p53 tetramerization domain [Wang,, et al. (1994), citedabove]. Analysis of the DNA binding activity of a deletion mutantlacking residues 353-360 indicates that it contains a functional NRR1(Table 1), which indicates that the N-terminal boundary of NRR1 is atresidue 360. Thus, the NRR1 is within residues 361-383 of p53.

Examination of mutants p53D300-308, p53D300-317 and p53D300-321 revealedthe unexpected presence of a second negative regulatory region (NRR2),since all these three deletion mutants exhibited activated binding tooligonucleotide BC.V4A. NRR2 does not overlap with the NRR1, since thetwo NRRs are separated by the p53 tetramerization domain (residues322-355 of p53 [Wang et al. (1994), cited above]).

TABLE 1 DNA Binding As Measured For Deletion Mutants of p53 Binding ofMutant Residues Deleted In Mutant Tested p53 to BC.V4A DNA None(wild-type p53) − 364-393 3+ 356-393 3+ 379-393 3+ 384-393 +/− 388-393+/− 353-360 +/− 300-308 3+ 300-317 3+ 300-321 3+

Example 4

Activation of p53 DNA Binding by Antibody PAb421

The p53 DNA binding assay involves incubating in vitro translated p53with radioactively-labeled DNAs containing p53 binding sites, such asoligonucleotides BC, BC.V4A. and Ewaf1 [see Example 2 above]. The effecton p53 DNA binding of incubation in the presence or absence of 0.1 μganti-p53 antibody PAb421 (available from Oncogene Science, Uniondale,N.Y.) is shown in Table 2. Wild-type p53 binds to oligonucleotides BC,BC.V4A and Ewaf1, as demonstrated by a slowly migrating 32P-labeledspecies, which represents the p53/DNA complex. In the presence ofantibody PAb421, the slowly migrating 32P-labeled species migrates evenmore slowly, indicating a greater size, since it now contains antibodyPAb421 as well. In addition, in the presence of PAb421, the intensity ofthe radioactivity emitted by the slowly migrating 32P-labeled speciesincreases, consistent with the observation that antibody PAb421activates DNA binding of wild-type p53 [Hupp et al. (1992), cited above;Halazonetis et al. (1993), cited above; Halazonetis and Kandil (1993),cited above). Because wild-type p53 binds quite efficiently tooligonucleotide BC, the ability of antibody PAb421 1lo activatewild-type p53 DNA binding is more evident with oligonucleotides BC.V4Aand Ewaf¹. Antibody PAb421 activates DNA binding of wild-type p53 byinactivating the negative regulatory region 1 (NRR1) at the p53C-terminus (see Background of the Invention).

Some of the p53 mutants frequently associated with human tumors (seeExample :2, above) were also examined for DNA binding usingoligonucleotide BC (see Table 2). Consistent with previous reports[Bargonetti et al. (1992), cited above], none of them exhibitedsignificant DNA binding activity as compared with wild-type p53.However, in the presence of antibody PAb421 mutants Gln248, His273,Cys273, Trp282 and Ser239 exhibited significant DNA binding activity(see Table 2), which for several of these mutants was equivalent to thatof wild-type p53.

TABLE 2 Stimulation of Mutant p53 Binding To BC DNA By Antibody SpecificFor NRR1 Amino Acid Substitution Stimulation of DNA Binding In p53Mutant By PAb421 None + Gln248 2+ His273 3+ Cys273 3+ Trp282 3+ Sep2383+

These experiments suggest that inactivation of the NRR1 by PAb421 canrestore DNA binding activity to some of the p53 mutants frequentlyassociated with human cancer. The peptides of this invention alsoinactivate the function of the NRR1 (although by a different mechanismthan PAb421), as shown in the following studies. Therefore, the spectrumof p53 mutants that are amenable to therapeutic intervention using thepeptides of this invention may be determined by identifying p53 mutantswhose DNA binding activity is susceptible to stimulation by PAb421.

Example 5

Activation of p53 DNA Binding by Peptides of the Invention

DNA binding assays were performed as described above (Example 2) in thepresence or absence of peptides corresponding to fragments of NRR1 fromhuman p53. The peptides examined are described in Example 1 above andwere tested at concentrations ranging between 0.02-0.4 mM. The resultsare summarized in Table 3.

We first examined the ability of peptide p53pC360-386 to activatebinding of wild-type p53 to oligonucleotide BC.V4A. In the absence ofthe peptide, wild-type p53 binds weakly lo this DNA. However, in thepresence of 0.02-0.2 mM peptide p53pC360-386, DNA binding of wild-typep53 is activated. The level of activation achieved by 0.02 mM peptidep53pC360-386 is similar to that achieved by 0.1 μg antibody PAb421. Theability of peptide p53pC360-386 to activate DNA binding of wild-type p53is not limited to oligonucleotide BC.V4A. DNA binding was also activatedto oligonucleotide Ewaf1 (see Table 3).

Since peptide p53pC360-386 activated DNA binding of wild-type p53, wesubsequently examined whether truncating this peptide at the N- orC-termini would maintain activity. Peptides p53p362-386, p53p363-386,p53p363-382 and p53p367-386 were examined in a DNA binding assay usingoligonucleotide Ewaf1, and the data is summarized in Table 3. At peptideconcentrations of 0.2-0.3 mM, all peptides activated DNA binding ofwild-type p53. An even smaller peptide, p53p371-383, was examined usingoligonucleotide BC.V4A. At peptide concentrations of 0.2-0.4 mM, peptidep53p371-383 activated DNA binding of wild-type p53.

We subsequently examined the ability of peptides p53p363-373,p53p368-380 and p53p373-383 to activate DNA binding of wild-type humanp53. These peptides correspond to consecutive partially overlappingregions of the p53 NRR1 (residues 363-373, 368-380 and 373-383 of humanp53, respectively). All three peptides activated binding of wild-typep53 to oligonucleotide Ewaf1, when tested at a 0.4 mM concentration.Peptides p53p363-373 and p53p368-380 were somewhat more potent thanp53p373-383 in activating DNA binding. Nevertheless, these resultssuggest that peptides corresponding to small regions of the p53 NRR1 canbe effective in activating p53 DNA binding. Furthermore, since peptidesp53p363-373 and p53p373-383 essentially don't overlap, the stimulatorypeptides need not contain a specific minimal fragment of the p53 NRR1.

To examine whether the peptides of this invention also activate DNAbinding of p53 mutants, we employed the His273 p53 mutant, as arepresentative of the class of mutants whose DNA binding can beactivated by inactivation of NRR1 (see Example 4 above). The p53His273mutant fails to bind to oligonucleotide BC using the DNA binding assaydescribed here. However, when incubated with peptides p53pC360-386 orp53p371-383 at 0.2 and 0.4 mM concentrations, respectively, thenp53His273 demonstrated DNA binding activity.

We conclude that the peptides of this invention activate DNA binding ofwild-type p.53 and of select p53 mutants as efficiently as antibodyPAb421. While both the peptides of this invention and antibody PAb421inactivate the NRR1 of p53, their mechanism of action is different.Thus, antibody PAb421 binds to NRR1 and inactivates it by masking it. Incontrast, the peptides of this invention compete with the endogenousNRR1 of p53 for binding to a second negative regulatory region, perhapsNRR2. Thus, they are competitors of NRR1, not masking agents.

TABLE 3 Extent of Stimulation by Peptides of p53 Binding to DNA Peptide(p53 residues) BC.V4A Ewaf1 Stimulation of wild-type p53 Binding to DNA360-386 3+ 3+ 362-386  ND* 3+ 363-386 ND 3+ 363-382 ND 3+ 367-386 ND 3+371-383 2+ ND 363-373 ND 3+ 368-380 ND 3+ 373-383 ND 2+ Stimulation ofDNA Binding by p53 Mutant His273 360-386 3+ ND 371-383 3+ ND *ND = NotDone

Example 6

Activation of p53 DNA Binding by a Cyclic Hexapeptide of the Invention

The following modified peptide is a cyclic peptide corresponding toresidues 378-382 of human p53. Its sequence is: Ser-Arg-His-Lys-Lys. Toallow this peptide to become cyclic, a D-alanine was introduced at itsC-terminus. This peptide is named c[SRHKKa] and its chemical structureis shown below:

Peptide c[SRHKKa] was examined in the p53 DNA binding assay described inExample 2. At a concentration of 0.1 mM this peptide activated p53 DNAbinding about two times more efficiently than 0.05 mM of peptidep53p363-373 [SEQ ID NO:4]. This example illustrates that: 1) five aminoacids of p53 sequence are sufficient to confer activity, and 2) thatcyclic peptides, which are not susceptible to N-terminal or C-terminalpeptidases, have activity, as claimed in our original application.

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in medicine,immunology, hybridoma technology, pharmacology, and/or related fieldsare intended to be within the scope of the invention, which is limitedonly by the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the compositions and processes of the present invention are believedto be encompassed in the scope of the claims appended hereto.

35 1 1317 DNA Homo sapiens 1 gtctagagcc accgtccagg gagcaggtag ctgctgggctccggggacac tttgcgttcg 60 ggctgggagc gtgctttcca cgacggtgac acgcttccctggattggcag ccagactgcc 120 ttccgggtca ctgccatgga ggagccgcag tcagatcctagcgtcgagcc ccctctgagt 180 caggaaacat tttcagacct atggaaacta cttcctgaaaacaacgttct gtcccccttg 240 ccgtcccaag caatggatga tttgatgctg tccccggacgatattgaaca atggttcact 300 gaagacccag gtccagatga agctcccaga atgccagaggctgctccccc cgtggcccct 360 gcaccagcag ctcctacacc ggcggcccct gcaccagccccctcctggcc cctgtcatct 420 tctgtccctt cccagaaaac ctaccagggc agctacggtttccgtctggg cttcttgcat 480 tctgggacag ccaagtctgt gacttgcacg tactcccctgccctcaacaa gatgttttgc 540 caactggcca agacctgccc tgtgcagctg tgggttgattccacaccccc gcccggcacc 600 cgcgtccgcg ccatggccat ctacaagcag tcacagcacatgacggaggt tgtgaggcgc 660 tgcccccacc atgagcgctg ctcagatagc gatggtctggcccctcctca gcatcttatc 720 cgagtggaag gaaatttgcg tgtggagtat ttggatgacagaaacacttt tcgacatagt 780 gtggtggtgc cctatgagcc gcctgaggtt ggctctgactgtaccaccat ccactacaac 840 tacatgtgta acagttcctg catgggcggc atgaaccggaggcccatcct caccatcatc 900 acactggaag actccagtgg taatctactg ggacggaacagctttgaggt gcgtgtttgt 960 gcctgtcctg ggagagaccg gcgcacagag gaagagaatctccgcaagaa aggggagcct 1020 caccacgagc tgcccccagg gagcactaag cgagcactgcccaacaacac cagctcctct 1080 ccccagccaa agaagaaacc actggatgga gaatatttcacccttcagat ccgtgggcgt 1140 gagcgcttcg agatgttccg agagctgaat gaggccttggaactcaagga tgcccaggct 1200 gggaaggagc caggggggag cagggctcac tccagccacctgaagtccaa aaagggtcag 1260 tctacctccc gccataaaaa actcatgttc aagacagaagggcctgactc agactga 1317 2 393 PRT Homo sapiens 2 Met Glu Glu Pro Gln SerAsp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser AspLeu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro Leu Pro SerGln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45 Asp Ile Glu Gln TrpPhe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50 55 60 Arg Met Pro Glu AlaAla Pro Pro Val Ala Pro Ala Pro Ala Ala Pro 65 70 75 80 Thr Pro Ala AlaPro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser 85 90 95 Val Pro Ser GlnLys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly 100 105 110 Phe Leu HisSer Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro 115 120 125 Ala LeuAsn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln 130 135 140 LeuTrp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met 145 150 155160 Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165170 175 Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln180 185 190 His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu AspAsp 195 200 205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu ProPro Glu 210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr MetCys Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro IleLeu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu GlyArg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly Arg AspArg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly Glu Pro HisHis Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala Leu Pro Asn AsnThr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp GlyGlu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325 330 335 Arg Phe Glu MetPhe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp 340 345 350 Ala Gln AlaGly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His 355 360 365 Leu LysSer Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met 370 375 380 PheLys Thr Glu Gly Pro Asp Ser Asp 385 390 3 390 PRT Mus spretus 3 Met ThrAla Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro 1 5 10 15 LeuSer Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro Glu 20 25 30 AspIle Leu Pro Ser Pro His Cys Met Asp Asp Leu Leu Leu Pro Gln 35 40 45 AspVal Glu Glu Phe Phe Glu Gly Pro Ser Glu Ala Leu Arg Val Ser 50 55 60 GlyAla Pro Ala Ala Gln Asp Pro Val Thr Glu Thr Pro Gly Pro Val 65 70 75 80Ala Pro Ala Pro Ala Thr Pro Trp Pro Leu Ser Ser Phe Val Pro Ser 85 90 95Gln Lys Thr Tyr Gln Gly Asn Tyr Gly Phe His Leu Gly Phe Leu Gln 100 105110 Ser Gly Thr Ala Lys Ser Val Met Cys Thr Tyr Ser Pro Pro Leu Asn 115120 125 Lys Leu Phe Cys Gln Leu Val Lys Thr Cys Pro Val Gln Leu Trp Val130 135 140 Ser Ala Thr Pro Pro Ala Gly Ser Arg Val Arg Ala Met Ala IleTyr 145 150 155 160 Lys Lys Ser Gln His Met Thr Glu Val Val Arg Arg CysPro His His 165 170 175 Glu Arg Cys Ser Asp Gly Asp Gly Leu Ala Pro ProGln His Leu Ile 180 185 190 Arg Val Glu Gly Asn Leu Tyr Pro Glu Tyr LeuGlu Asp Arg Gln Thr 195 200 205 Phe Arg His Ser Val Val Val Pro Tyr GluPro Pro Glu Ala Gly Ser 210 215 220 Glu Tyr Thr Thr Ile His Tyr Lys TyrMet Cys Asn Ser Ser Cys Met 225 230 235 240 Gly Gly Met Asn Arg Arg ProIle Leu Thr Ile Ile Thr Leu Glu Asp 245 250 255 Ser Ser Gly Asn Leu LeuGly Arg Asp Ser Phe Glu Val Arg Val Cys 260 265 270 Ala Cys Pro Gly ArgAsp Arg Arg Thr Glu Glu Glu Asn Phe Arg Lys 275 280 285 Lys Glu Val LeuCys Pro Glu Leu Pro Pro Gly Ser Ala Lys Arg Ala 290 295 300 Leu Pro ThrCys Thr Ser Ala Ser Pro Pro Gln Lys Lys Lys Pro Leu 305 310 315 320 AspGly Glu Tyr Phe Thr Leu Lys Ile Arg Gly Arg Lys Arg Phe Glu 325 330 335Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala His Ala 340 345350 Thr Glu Glu Ser Gly Asp Ser Arg Ala His Ser Ser Tyr Leu Lys Thr 355360 365 Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Thr Met Val Lys Lys370 375 380 Val Gly Pro Asp Ser Asp 385 390 4 11 PRT Homo sapiens 4 ArgAla His Ser Ser His Leu Lys Ser Lys Lys 1 5 10 5 13 PRT Homo sapiens 5His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His 1 5 10 6 11 PRT Homosapiens 6 Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu 1 5 10 7 13 PRTHomo sapiens 7 Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu 1 510 8 20 PRT Homo sapiens 8 Arg Ala His Ser Ser His Leu Lys Ser Lys LysGly Gln Ser Thr Ser 1 5 10 15 Arg His Lys Lys 20 9 20 PRT Homo sapiens 9Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys 1 5 1015 Leu Met Phe Lys 20 10 24 PRT Homo sapiens 10 Arg Ala His Ser Ser HisLeu Lys Ser Lys Lys Gly Gln Ser Thr Ser 1 5 10 15 Arg His Lys Lys LeuMet Phe Lys 20 11 25 PRT Homo sapiens 11 Ser Arg Ala His Ser Ser His LeuLys Ser Lys Lys Gly Gln Ser Thr 1 5 10 15 Ser Arg His Lys Lys Leu MetPhe Lys 20 25 12 27 PRT Homo sapiens 12 Gly Gly Ser Arg Ala His Ser SerHis Leu Lys Ser Lys Lys Gly Gln 1 5 10 15 Ser Thr Ser Arg His Lys LysLeu Met Phe Lys 20 25 13 11 PRT Artificial Sequence Synthetic, modifiedfrom Homo sapiens p53 13 Lys Lys Ser Lys Leu His Ser Ser His Ala Arg 1 510 14 8 PRT Homo sapiens 14 Arg Ala His Ser Ser His Leu Lys 1 5 15 6 PRTHomo sapiens 15 His Leu Lys Ser Lys Lys 1 5 16 5 PRT Homo sapiens 16 HisLeu Lys Ser Lys 1 5 17 5 PRT Homo sapiens 17 Leu Lys Ser Lys Lys 1 5 186 PRT Homo sapiens 18 Lys Ser Lys Lys Gly Gln 1 5 19 5 PRT Homo sapiens19 Lys Ser Lys Lys Gly 1 5 20 7 PRT Artificial Sequence synthetic,modified from human p53 20 Arg Ala His Ser His Leu Lys 1 5 21 5 PRTArtificial Sequence synthetic, modified from human p53 21 His Lys SerLys Lys 1 5 22 28 PRT Artificial Sequence synthetic, modified from humanp53 22 Cys Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly 15 10 15 Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys 20 25 23 27 PRTArtificial Sequence synthetic, modified from human p53 23 Cys Gly SerArg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln 1 5 10 15 Ser ThrSer Arg His Lys Lys Leu Met Phe Lys 20 25 24 27 PRT Artificial Sequencesynthetic, modified from human p53 24 Cys Gly Gly Ser Arg Ala His SerSer His Leu Lys Ser Lys Lys Gly 1 5 10 15 Gln Ser Thr Ser Arg His LysLys Leu Met Lys 20 25 25 26 PRT Artificial Sequence synthetic, modifiedfrom human p53 25 Cys Gly Ser Arg Ala His Ser Ser His Leu Lys Ser LysLys Gly Gln 1 5 10 15 Ser Thr Ser Arg His Lys Lys Leu Met Lys 20 25 261215 DNA Homo sapiens 26 gaattcaacc agcagcctcc cgcgaccatg gaggagccgcagtcagatcc tagcgtcgag 60 ccccctctga gtcaggaaac attttcagac ctatggaaactacttcctga aaacaacgtt 120 ctgtccccct tgccgtccca agcaatggat gatttgatgctgtccccgga cgatattgaa 180 caatggttca ctgaagaccc aggtccagat gaagctcccagaatgccaga ggctgctccc 240 cccgtggccc ctgcaccagc agctcctaca ccggccgcccctgcaccagc cccctcctgg 300 cccctgtcat cttctgtccc ttcccagaaa acctaccagggcagctacgg tttccgtctg 360 ggcttcttgc attctgggac agccaagtct gtgacttgcacgtactcccc tgccctcaac 420 aagatgtttt gccaactggc gaagacctgc cctgtgcagctgtgggttga ttccacaccc 480 ccgcccggca cccgcgtccg cgccatggcc atctacaagcagtcacagca catgacggag 540 gttgtgaggc gctgccccca ccatgagcgc tgctcagatagcgatggtct ggcccctcct 600 cagcatctta tccgagtgga aggaaatttg cgtgtggagtatttggatga cagaaacact 660 tttcgacata gtgtggtggt accctatgag ccgcctgaggttggctctga ctgtaccacc 720 atccactaca actacatgtg taacagttcc tgcatgggcggcatgaaccg gaggcccatc 780 ctcaccatca tcacactgga agactccagt ggtaatctactgggacggaa cagctttgag 840 gtgcgtgttt gtgcctgtcc tgggagagac cggcgcacagaggaagagaa tctccgcaag 900 aaaggggagc ctcaccacga gctcccccca gggagcactaagcgagcact gcccaacaac 960 accagctcct ctccccagcc aaagaagaaa ccactggatggagaatattt cacccttcag 1020 atccgcgggc gtgagcgctt cgaaatgttc cgagagctgaatgaggcctt ggaactcaag 1080 gatgcccagg ctgggaagga gccagggggg agcagggctcactccagcca cctgaagtcc 1140 aaaaagggtc agtctacctc ccgccataaa aaactcatgttcaagacaga agggcctgac 1200 tcagactgag tcgac 1215 27 27 DNA Homo sapiens27 gagagcccca gttaccataa ctactct 27 28 30 DNA Homo sapiens 28 atcacgtgatatcacgtgat atcacgtgat 30 29 26 DNA Homo sapiens 29 cccgaacatg tcccaacatgttgggg 26 30 30 DNA Homo sapiens 30 tcgagcatgt tcgagcatgt tcgagcatgt 3031 30 DNA Homo sapiens 31 ccgggcatgt ccgggcatgt ccgggcatgt 30 32 5 PRTHomo sapiens 32 Lys Ser Lys Lys Gln 1 5 33 8 PRT Artificial Sequencesynthetic, modified from human p53 33 Arg Ala His Ser Ser His Lys Lys 15 34 6 PRT Artificial Sequence synthetic, modified from human p53 34 HisLeu Lys Ser Arg His 1 5 35 20 DNA Homo sapiens 35 tggcatgtca tggcatgtca20

What is claimed is:
 1. A method for identifying a peptidomimetic whichactivates DNA binding of wild-type p53, said method comprising the stepsof selecting a non-peptidyl compound of a peptide containing at least 4amino acids from a negative regulatory region which maps to residues361-383 of p53, and determining the ability of said compound tostimulate DNA binding activity in a p53 DNA binding assay.
 2. Apeptidomimetic of a peptide identified according to claim 1, whereinsaid peptide competes with an endogenous p53 negative regulatory regionfor binding to a negative regulatory region of p53, said peptidecontaining at least four sequential amino acids from a negativeregulatory region which maps to residues 361-383 (SEQ ID NO. 2) of p53,said peptide not being a subfragment of human p53, wherein said peptideactivates, in a p53 DNA binding assay, DNA binding of wild-type p53 or ap53 mutant containing a single amino acid substitution, said mutantselected from the group consisting of p53-ser²³⁹, p53-his²⁷³,p53-gln²⁴⁸, p53-trp²⁸², and p53-cys²⁷³, and wherein said peptidomimeticstimulates DNA binding activity in said p53 DNA binding assay.
 3. Thepeptidomimetic of claim 2, comprising at least four sequential aminoacids which map to residues 361-383 (SEQ ID NO. 2) of p53, wherein saidpeptidomimetic comprises one or more peptide bonds; and wherein one ormore said peptide bonds are replaced by a reduced isostere pseudopeptidebond.
 4. The peptidomimetic of claim 2, wherein said peptidomimeticcomprising one or more peptide bonds, and wherein one or more saidpeptide bonds are replaced by a retro-inverso pseudopeptide bond.
 5. Thepeptidomimetic of claim 2, wherein one or more said amino acids arereplaced by an N-substituted glycine.
 6. A composition comprising saidpeptidomimetic of a peptide of claim 2 and a pharmaceutically acceptablecarrier.
 7. The method of claim 1, wherein said peptide containsresidues selected from the group consisting of p53-ser²³⁹, p53-his²⁷³,p53-gln²⁴⁸, p53-trp²⁸², and p53-cys²⁷³, 363-370 of human p53, 368-372 ofhuman p53, 369-373 of human p53, 370-374 of human p53, and 378-382 ofhuman p53.
 8. The method of claim 1, wherein at least one of said atleast 4 amino acids is substituted by a corresponding residue from anon-human p53 sequence.
 9. The method of claim 1, wherein said peptidecontains both L-amino acids and D-amino acids.
 10. The method of claim9, wherein said peptide contains an N-terminal amino acid which is aD-amino acid.
 11. The method of claim 9, wherein said peptide contains aC-terminal amino acid which is a D-amino acids.
 12. The method of claim1, wherein said at least 4 amino acids are D-amino acids which map toresidues 361-383 of human p53 in a reverse sequence orientation relativeto wild-type human p53.
 13. The method of claim 1, wherein said peptideis cyclic.
 14. The method of claim 1, wherein one or more natural aminoacids of said peptide are substituted by an unnatural amino acid. 15.The method of claim 14, wherein said natural amino acid is serine andsaid unnatural amino acid is isoserine.
 16. A method for identifying apeptidomimetic which activates DNA binding of wild-type p53, said methodcomprising the steps of selecting a non-peptidyl compound of a peptidecontaining at least 4 amino acids from a negative regulatory regionwhich maps to residues 361-383 of p53, said peptide not being asubfragment of human p53 and determining the ability of said compound tostimulate DNA binding activity in a p53 DNA binding assay.
 17. Thepeptidomimetic of claim 2, wherein one or more peptide bonds arereplaced by a reduced isostere pseudopeptide bond.